The innate immunity, a defense system which is common to vertebrates and invertebrates, is based on the recognition of molecules of microbial origin, known as Pathogen-Associated Molecular Patterns or PAMPs (Pathogen-Associated Molecular Patterns) and molecules of endogenous origin, named HAMPs (Host-Associated Molecular Patterns) or DAMPs (Damage-Associated Molecular Patterns), which indicate the presence of potential pathogens.
In plants, an example of DAMPs is represented by oligogalacturonides (OG), molecules derived from the degradation of the pectic component of the wall, due to polygalacturonases released by pathogens micro-organisms during invasion.
OGs function as danger signals and induce the expression of defense genes and proteins (Ridley 2001, Denoux 2008, Casasoli 2008, Galletti 2008, Casasoli 2009), protecting plants against fungal diseases (Ferrari 2007). Besides inducing defense responses, OGs also affect several aspects of plant growth and development (Ridley, 2001, Bellincampi 1996). Both at the structural and the functional level, OGs are reminiscent of the hyaluronan fragments of the animal extracellular matrix, a well known class of DAMPs involved in wound response and healing (Jiang, 2007). Like that of hyaluronan fragments, biological activity of OGs is related to their molecular size, since fragments with a degree of polymerization (DP) comprised between 10 and 15 are the most active (13, 14).
Notably, the “egg box” conformation is necessary for the biological activity of OGs (Ridley, 2001, Cabrera 2008).
The more studied PAMPs are the bacterial flagellin and the elongation factor Tu (EF-Tu) that, in Arabidopsis, are perceived by two LRR receptor kinase (LRR-RLK, Leucine-Rich Repeat Receptor-Like Kinase), called FLS2 and EFR, respectively. These proteins are analogous to human TLR receptors (Toll-Like Receptor) and consist of an extracellular LRR domain, a single-stranded transmembrane region and an intracellular kinase domain of the serine/threonine type. The recognition of the ligand in the two systems, flagellin/FLS2 and EF-Tu/EFR, determines the activation of complex defense responses, largely shared, like the expression of genes involved in defense responses, the accumulation of ethylene, callose, hydrogen peroxide and finally the induction of hypersensitivity response. It is also known that the EFR receptor, after the recognition of its ligand, activates these defense responses not only in Arabidopsis but in other plant species, too (Lacombe, 2010).
There are many phytopathogens of different origin, such as viral, bacterial and fungal that can significantly reduce the productivity of crops, causing lesions in the plant tissues, reducing the development of leaves, roots or seeds. In absence of obvious symptoms, pathogens can cause a general metabolic disorder that reduces the productivity of the plants themselves. Pathogens can cause damage to pre- or post-harvest. Strategies for chemical control of diseases have obvious disadvantages, due to high costs and occasional toxicity for the non-target organisms.
Cell wall is the extracellular matrix that separates the plant cell from the external environment and plays a fundamental role in filtering and interpreting external cues such as pathogen attack, wounding or mechanical stress (Kohorn 2000; Brownlee 2002). Pectin, a component of the cell wall that is continually modified and remodelled during plant growth and development, is a complex polymer that determines the porosity, hydration and plasticity of the wall as well as cell-cell adhesion. Moreover, pectin is critical for physiological processes such as pollen growth (Stenzel et al., 2008) and compatibility (Lord 2003), root and stem elongation, seed germination and fruit ripening (Micheli 2001; Pilling et al., 2004) as well as for response to pathogens not just as a mechanical barrier but also as a sensor for incoming infections (Vorwerk et al., 2004); The characteristic of pectin that determines maintenance of the wall integrity and cohesion of the cells is due to the polyanionic nature of its backbone, i.e. homogalacturonan, which is capable of binding calcium to form the structures called “egg-box”. These structures can occasionally be hydrolysed and fragmented by enzymes of microbial or vegetal origin, to release the OG which perform regulative and activation actions of defense responses (Cervone et al., 1989). Treatment of plant tissues with OGs causes accumulation of reactive oxygen species, biosynthesis of phytoalexins and expression of pathogenesis-related (PR) proteins (Ridley et al., 2001). In Arabidopsis OGs induce the expression of genes and defense proteins (Denoux et al., 2008; Casasoli et al., 2008) and protect the plant against fungal diseases (Ferrari et al., 2007). In analogy with the role of hyaluronan fragments in the animal innate immunity, OGs may be regarded as host-associated molecular patterns (HAMPS); (Taylor & Gallo 2006; Stern et al., 2006)). Besides inducing defense responses, OGs also affect several aspects of plant growth and development (Bellincampi et al., 1996; Mauro et al., 2002).
Since the response of Arabidopsis to OGs largely overlaps that to PAMPs flg22 (peptide derived from bacterial flagellin) (Denoux et al., 2008) and elft 8 (peptide derived from EF-Tu (Zipfel et al., 2006) it has been hypothesized that the receptor of OGs is similar to the receptors Flagellin Sensing 2 (FLS2) and Elongation Factor Tu Receptor (EFR). These are members of the leucine-rich repeat (LRR) receptor kinase (RK) family (Zipfel 2008; Sanabria et al., 2008) and the observation that an extracellular LRR protein, i.e. the polygalacturonase-inhibiting protein PGIP, interacts with OGs supports this hypothesis (Spadoni et al., 2006). On the other hand, candidate receptors of OGs are also some members of the Wall-Associated Kinase (WAK) family.
The WAK proteins are kinase-proteins belonging to the RLK (Receptor Like Kinase) family, showing an intracellular kinase domain of the Ser/Thr type and an extracellular domain containing multiple repeats, similar to epidermal growth factor (EGF) (He et al., 1996). In Arabidopsis, there are five genes that are tightly packed and highly correlated (WAK1-WAK5); they are expressed in leaves and meristems subjected to expansion, and are induced by pathogens, wounding and mechanical stress (He et al. 1996; Verica et al., 2003). The WAK family correlates with another family called WAK-like which includes 22 members (Verica & He 2002). WAK1 (At1g21250), the best characterized gene, is highly expressed in green organs and is induced by salicylic acid, and encodes a mature protein of 711 amino acids (He et al., 1998). WAK1 binds in vitro to the non-methyl esterificated homogalacturonan, to the OG with a degree of polymerization between 9 and 14 active as elicitor and compatible with the formation of “egg-box” structures calcium-induced (Decreux & Messiaen 2005, Cabrera et al., 2008). The antisense and inducible expression of WAK2 or WAK4 causes a reduction of WAK protein levels and a dwarf phenotype (Wagner & Kohorn 2001, Lally et al., 2001). The knock out mutant wak2 showed a dependence on sugar (and salt) for sprout's growth, suggesting that the WAK proteins are involved in the regulation of sugar metabolism (Kohorn et al., 2006b).
However, the precise role of individual WAK receptors remains largely unknown (Decreux & Messiaen 2005).
In Arabidopsis, only a minimal number of the over 600 RLKs have been characterized (Shiu et al., 2004; Afzal et al., 2008) and the possible occurrence within this superfamily of multiple members with similar and redundant function makes a reverse genetic approach (gene knock-out) for the identification of the receptors perceiving a pleiotropic signal like OGs, very difficult.
The construction of chimeric receptors, constituted by domains of different proteins, is an alternative approach for the biochemical and functional characterization of RLKs. Domain swaps have been widely used to study animal receptors (Tauszig et al., 2000; Tsujita et al., 2004; Weber et al., 2005), while in Arabidopsis it has been reported only one example in which the LRR ectodomain of the receptor kinase BRI1 is fused to the serine/threonine kinase domain of the rice gene product Xa21 and is able to initiate plant defense responses in rice cells upon treatment with brassinosteroids (He et al., 2000). Using a similar design, the ectodomain-TM-iJM portion of the rice resistance gene Xa3/Xa26 was fused to the kinase domains of either MRKa or MRKc, which belong to the same gene family as Xa3/Xa26 and expressed in rice. The transgenic plants were reported to be partially resistant to Xanthomonas oryzae pv. oryzae (Cao et al., 2007b).